This R15 AREA application focuses on the development of a new bioprocess sensing technology poised to enhance biomedical research and training within the New Mexico State University department of chemical engineering. A new diagnostic system is under development for the rapid advancement of temporally-sensitive flow cytometry techniques. That is, new methodologies are proposed for the measurement of heterogeneous excited state fluorescence decays, near- instantaneous inelastic scattering rates, fluorescence lifetime, and phase-filtering approaches from cells and particles in transient states. The proposed time resolved techniques are investigated for real-time cytometric analysis as well as sorting. In addition, these new technologies are developed to identify rare decay kinetic phenomena from individual cells and/or particles, to explore emerging cytometry applications, and for investigation of was to improve and address common cytometry gaps such as autofluorescence noise-plagued assays. To support the development of heterogenous excited-state sorting and analysis cytometry (HESAC), two hypotheses will be examined: (1) that high-throughput multi-exponential fluorescence decay measurements of endogenous cellular emission will not only separate bulk autofluorescence from weak exogenous emissions but also reveal discrete intrinsic species, expose rare events related to intrinsic proteins present at different cell cycles and states, permit separation of Raman scatter for multiplexing, and indicate energy transfer effects;and (2) that the direct selection and screening of stable fluorescent proteins, which are surmised to have distinct heterogeneous lifetimes based on thermally induced conformational changes (i.e. unfolding) and vibrational disruptions (immediate environmental changes), will benefit from fluorescence lifetime-based sorting. The technology development and research questions to be studied in this application have a major potential for widespread influence on the larger cytometry community. Significant contributions to cytometry research are anticipated owing to technological characteristics such as (i) compactness, for point-of-care diagnostics, (ii) multifaceted, for facile commercial integration, (iii) expandable, for development into biomedical molecular imaging devices and systems;and (iv) sophisticated, for the exploration of emerging cytometry assays and never-before studied phenomena. It is projected that the study of high-impact cytometry research will progress into further questions related to sorting, and analysis of cells and particles. Ultimately the most important contribution is that New Mexico State University undergraduate and graduate students be exposed to novel biomedical engineering concepts, methodologies, and principles that coalesce with discoveries in biology, chemistry, and physics disciplines, and that lead to the commercial introduction of quantitative heterogeneous excited-state decay analysis and sorting cytometry. PUBLIC HEALTH RELEVANCE: Development and validation of heterogeneous excited-state-dependent sorting and analysis cytometry (HESAC) for biomedical research applications, clinical diagnostics, and use in point-of-care cytometry systems.